For many years, various cell culture systems have been used in preclinical drug development. However, established cell models only partially reflect pharmaceutically relevant disease-specific physiology because they are either derived from tumorigenic tissue or from transformed and immortalized cells. In particular, because terminally-differentiated cardiomyocytes have been shown to possess limited proliferative potential, they do not have the capacity to effectively generate cell models for drug development. Hence there is a need for more disease relevant human cell types that can be used as reliable cell models in research and drug development.
Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) provide researchers with immense opportunities for generating functional human cell types such as cardiomyocytes, neuronal cells, pancreatic cells, etc. Robust protocols for in vitro differentiation of pure hESC- and iPSC-derived human cardiomyocyte (hESCM) cultures would present a powerful tool, not only to advance the understanding of early human cardiogenesis, but also to use the cardiomyocytes as a non-transformed human cell model to test drug efficacy in preclinical stages of drug development and to assess cardiac toxicity before entering the clinic. Additionally, hESC-derived human cardiomyocytes could open opportunities for identifying pathways critical to cardiac regeneration and ultimately lead to clinical applications supporting stem-cell based therapy.
For developing cell assay models for pharmaceutical research and development, such differential protocols need to generate cells that ideally fulfill the following criteria: a) are robust with a high level of reproducibility; b) generate large numbers of highly pure cell types; c) can be differentiated in a short time; d) generate cells that can be frozen to ensure batch conformity for multiple screening campaigns; e) provide functionality and physiology relevant for modeling disease-specific readouts. (For a review of prior art approaches to differentiate pluripotent cells into cardiomyocytes, see Burridge et al. (2012) Cell Stem Cell 10:16-28.) So far none of the known protocols fulfil the criteria above. In particular, cardiomyocytes obtained through the known protocols are difficult to freeze and thaw without losing any functional properties.
To fulfill these requirements, the instant inventors developed a novel differentiation method that generates large numbers of highly pure cardiomyocytes (up-to 95%). The differentiation protocol makes use of defined small molecules to direct differentiation towards the cardiac lineage in a time span of 10 days. To further increase their purity, the cardiomyocytes are enriched by replating them using conditions that are preferential for cardiomyocytes. Furthermore, the cardiomyocytes can afterwards be frozen, stored under liquid nitrogen, and thawed again. The cardiomyocytes have been tested to be compliant with several screening formats used in pharmaceutical research and development. The present invention provides an improved method for differentiating pluripotent stem cells into cardiomyocytes in a shorter amount of time and with a significantly increased yield compared to prior art protocols. The new method alleviates the necessity of obtaining embryoid bodies or small cell clumps from pluripotent stem cells and removes the major drawback of low reproducibility and standardization of methods known so far. Moreover, the high efficiency allows the use of these defined cardiomyocytes in large scales in drug discovery and safety assessments, in regenerative medicine applications, and in in vitro disease modeling in the pharmaceutical industry.